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n more genotypes pres ent in the data than female lines, This is the only variant region for PP1 P. zelicaon with more than 10 variants. B Parameter Because ESTs were sequenced from a number of geno types and because assembly coverage varies among con tigs, standard measures of nucleotide diversity such as θ can not PP1 be calculated. Instead, we consider a relative measure of nucleotide diversity Bt developed by Novaes et al, defined for contigs with average coverage at least 2×. However, for all that follows, we compute B statis tics only for those contigs with at least 6× average cover age to avoid biases caused by contigs that represent diverse sequences but are expressed at low levels. For contigs that also have a B.
mori best hit, we can compute RGFP966 Bn, a diversity estimate for non synony mous sites, and Bs, a diversity estimate for synonymous sites, Bt, Bn, and Bs are formally defined as follows. In the above, St is the number RNA polymerase of SNPs in the contig, Sn is the number of non synonymous SNPs in BLAST annotated putative coding regions, Ss is the number of synonymous SNPs in putative coding regions, Lt is the total length of the contig, Lc is the length of the putative coding region, D is the average coverage depth, and Hn is the nth har monic number. Table 4 shows average and median values of Bt, Bn and Bs amongst contigs with at least 6× coverage for both species. Novaes et al. note that because B statistics are condi tioned on coverage depth rather than the actual number of haplotypes sampled, care must be taken in comparing to more traditional diversity estimates such as θ, However, these statistics do enable the study of relative genetic diversity within each transcriptome, and may speak to comparative diversity estimates for E.
propertius RGFP966 PP1 and P. zelicaon if allele sample rates are equal, The average coverage for E. propertius contigs in the top 1% of Bt was relatively low at 8. 8×, The average Bt for E. propertius con tigs in the top 1% of coverage also was low at 0. 68 × 10 3. For P. zelicaon, average coverage in the top 1% of Bt was 10. 64×, and the average Bt in the top 1% of coverage was 1. 29 × 10 3. Thus, for both species, very diverse contigs tend to have less than or near average coverage. conversely, highly cov ered contigs have low diversity. In the presence of large scale paralog collapse, we would expect to see many con tigs with high coverage and high B, which we have not found.
Discussion For E. propertius, the large RGFP966 sequences produced by the 454 FLX Titanium allowed for the formation of a 14. 6 Mbp assembly from 176 Mbp of EST PP1 sequence, with aver age contig coverage of 10× and average contig length of 753 bp. Similar results were obtained for P. zelicaon. Comparisons to Bombyx mori suggest that our final assemblies are of high quality. Because Bt was generally low for highly covered contigs, and nearly all variant regions had fewer variants than the number of genotypes sequenced, we see little evidence for over assembly and paralog collapse. Further, the fact that amongst the assemblies tested we have not seen a point of diminishing returns in terms of average ortholog hit ratio suggests that even more aggressive assemblers may pro duce more accurate assemblies for such diverse datasets.
Clustering results and comparison to the P. xuthus mitochondrial genome indicate the presence of ribo somal RNA in at least the P. zelicaon dataset. Although mitochondrial genes are polyadeny lated and appropriately found in our datasets, ribosomal RNAs are not, and hence should be considered contami nation. RGFP966 While such unigenes can easily be filtered after assembly, the fact that many of these were clustered via hits to a protein predicted dataset highlights the need for well annotated and curated reference data sets. Clustering results also reveal that greater than 90% of unigenes had no similarity with other unigenes, indicat ing thorough assemblies. We searched for five single copy genes present in B. mori, For those E. propertiu